A TAILOR-MADE PEPTIDE-AFFINITY COLUMN TO IDENTIFY NOVEL BINDING PARTNERS FOR A CYTOPLASMIC MOTIF OF THE PLATELET INTEGRIN ALPHA IIB
Abstract number: P-T-275
Raab1 M., Daxecker1 H., Devocelle2 M., Treumann1 A., Moran1 N.
11Molecular and Cellular Therapeutics 22Department of Chemistry, Royal College of Surgeons in Ireland, Dublin, Ireland
How-to-cite Raab M, Daxecker H, Devocelle M, Treumann A, Moran N. A TAILOR-MADE PEPTIDE-AFFINITY COLUMN TO IDENTIFY NOVEL BINDING PARTNERS FOR A CYTOPLASMIC MOTIF OF THE PLATELET INTEGRIN ALPHA IIB. J Thromb Haemost 2007; 5 Supplement 2: P-T-275
Introduction: Platelet aggregation, adhesion and spreading depends on the function of the abundant platelet-specific integrin alpha IIb beta 3. The highly-conserved KVGFFKR cytoplasmic motif of the integrin alpha IIb-subunit critically regulates the activation state of this integrin, probably by recruiting and interacting with signaling proteins. To date six platelet-proteins have been identified that bind to this motif or a stretch thereof: ICln, CIB, PP1, AUP-1, IKAP-1 and TIM.
Methods: In order to identify novel integrin binding partners in platelets we have developed and optimized a peptide-affinity chromatography method utilizing Toyopearl AF-Amino-650M resin presenting an immobilized peptide, LAMWKVGFFKR, as a bait.
Results: We show that specific binding of positive-control protein-standards (ICln, PP1, CIB, IKAP-1) to this peptide is achieved employing the following mobile phase system: 10 mM HEPES, 400 mM NaCl, pH 7 at 0.3 ml/min flow. Bound proteins are then selectively eluted from the stationary phase using 6 M guanidinium-HCl.
Injecting a lysate of gel-filtered platelets into this chromatographic system resulted in the retention of specific binding partners. The binding of ICln, CIB, PP1, IKAP-1, TIM and beta 3-integrin to the alpha-integrin regulatory peptide sequence was confirmed by SDS-PAGE, western- and dot-blotting, while novel integrin binding partners were identified by LC-MS/MS analysis.
Conclusions: This highly reproducible method allows the reliable extraction of specific binding proteins from complex biological mixtures like platelet lysates within run times of 90 minutes. Therefore it has the potential to serve as an optimal tool to identify novel integrin binding partners in resting and activated platelets, thus, improving the understanding of integrin regulation.
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Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number
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